Takeshige Kunieda

G-CSF prevents cardiac remodeling after myocardial infarction by activating the Jak-Stat pathway in cardiomyocytes

Granulocyte colony-stimulating factor (G-CSF) was reported to induce myocardial regeneration by promoting mobilization of bone marrow stem cells to the injured heart after myocardial infarction, but the precise mechanisms of the beneficial effects of G-CSF are not fully understood. Here we show that G-CSF acts directly on cardiomyocytes and promotes their survival after myocardial infarction. G-CSF receptor was expressed on cardiomyocytes and G-CSF activated the Jak/Stat pathway in cardiomyocytes. The G-CSF treatment did not affect initial infarct size at 3 d but improved cardiac function as early as 1 week after myocardial infarction. Moreover, the beneficial effects of G-CSF on cardiac function were reduced by delayed start of the treatment. G-CSF induced antiapoptotic proteins and inhibited apoptotic death of cardiomyocytes in the infarcted hearts. G-CSF also reduced apoptosis of endothelial cells and increased vascularization in the infarcted hearts, further protecting against ischemic injury. All these effects of G-CSF on infarcted hearts were abolished by overexpression of a dominant-negative mutant Stat3 protein in cardiomyocytes. These results suggest that G-CSF promotes survival of cardiac myocytes and prevents left ventricular remodeling after myocardial infarction through the functional communication between cardiomyocytes and noncardiomyocytes.

Akt negatively regulates the in vitro lifespan of human endothelial cells via a p53/p21-dependent pathway.

The signaling pathway of insulin/insulin‐like growth factor‐1/phosphatidylinositol‐3 kinase/Akt is known to regulate longevity as well as resistance to oxidative stress in the nematode Caenorhabditis elegans. This regulatory process involves the activity of DAF‐16, a forkhead transcription factor. Although reduction‐of‐function mutations in components of this pathway have been shown to extend the lifespan in organisms ranging from yeast to mice, activation of Akt has been reported to promote proliferation and survival of mammalian cells. Here we show that Akt activity increases along with cellular senescence and that inhibition of Akt extends the lifespan of primary cultured human endothelial cells. Constitutive activation of Akt promotes senescence‐like arrest of cell growth via a p53/p21‐dependent pathway, and inhibition of forkhead transcription factor FOXO3a by Akt is essential for this growth arrest to occur. FOXO3a influences p53 activity by regulating the level of reactive oxygen species. These findings reveal a novel role of Akt in regulating the cellular lifespan and suggest that the mechanism of longevity is conserved in primary cultured human cells and that Akt‐induced senescence may be involved in vascular pathophysiology.

Obesity in mice with adipocyte-specific deletion of clock component Arntl

Adipocytes store excess energy in the form of triglycerides and signal the levels of stored energy to the brain. Here we show that adipocyte-specific deletion of Arntl (also known as Bmal1), a gene encoding a core molecular clock component, results in obesity in mice with a shift in the diurnal rhythm of food intake, a result that is not seen when the gene is disrupted in hepatocytes or pancreatic islets. Changes in the expression of hypothalamic neuropeptides that regulate appetite are consistent with feedback from the adipocyte to the central nervous system to time feeding behavior. Ablation of the adipocyte clock is associated with a reduced number of polyunsaturated fatty acids in adipocyte triglycerides. This difference between mutant and wild-type mice is reflected in the circulating concentrations of polyunsaturated fatty acids and nonesterified polyunsaturated fatty acids in hypothalamic neurons that regulate food intake. Thus, this study reveals a role for the adipocyte clock in the temporal organization of energy regulation, highlights timing as a modulator of the adipocyte-hypothalamic axis and shows the impact of timing of food intake on body weight.

Angiotensin II Induces Premature Senescence of Vascular Smooth Muscle Cells and Accelerates the Development of Atherosclerosis via a p21-Dependent Pathway

Background— Angiotensin II (Ang II) has been reported to contribute to the pathogenesis of various human diseases including atherosclerosis, and inhibition of Ang II activity has been shown to reduce the morbidity and mortality of cardiovascular diseases. We have previously demonstrated that vascular cell senescence contributes to the pathogenesis of atherosclerosis; however, the effects of Ang II on vascular cell senescence have not been examined. Methods and Results— Ang II significantly induced premature senescence of human vascular smooth muscle cells (VSMCs) via the p53/p21-dependent pathway in vitro. Inhibition of this pathway effectively suppressed induction of proinflammatory cytokines and premature senescence of VSMCs by Ang II. Ang II also significantly increased the number of senescent VSMCs and induced the expression of proinflammatory molecules and of p21 in a mouse model of atherosclerosis. Loss of p21 markedly ameliorated the induction of proinflammatory molecules by Ang II, thereby preventing the development of atherosclerosis. Replacement of p21-deficient bone marrow cells with wild-type cells had little influence on the protective effect of p21 deficiency against the progression of atherogenesis induced by Ang II. Conclusions— We demonstrated that Ang II promotes vascular inflammation by inducing premature senescence of VSMCs both in vitro and in vivo. Our results suggest a critical role of p21-dependent premature senescence of VSMCs in the pathogenesis of atherosclerosis.

Critical Roles of Muscle-Secreted Angiogenic Factors in Therapeutic Neovascularization

The discovery of bone marrow–derived endothelial progenitors in the peripheral blood has promoted intensive studies on the potential of cell therapy for various human diseases. Accumulating evidence has suggested that implantation of bone marrow mononuclear cells effectively promotes neovascularization in ischemic tissues. It has also been reported that the implanted cells are incorporated not only into the newly formed vessels but also secrete angiogenic factors. However, the mechanism by which cell therapy improves tissue ischemia remains obscure. We enrolled 29 “no-option” patients with critical limb ischemia and treated ischemic limbs by implantation of peripheral mononuclear cells. Cell therapy using peripheral mononuclear cells was very effective for the treatment of limb ischemia, and its efficacy was associated with increases in the plasma levels of angiogenic factors, in particular interleukin-1&bgr; (IL-1&bgr;). We then examined an experimental model of limb ischemia using IL-1&bgr;–deficient mice. Implantation of IL-1&bgr;–deficient mononuclear cells improved tissue ischemia as efficiently as that of wild-type cells. Both wild-type and IL-1&bgr;–deficient mononuclear cells increased expression of IL-1&bgr; and thus induced angiogenic factors in muscle cells of ischemic limbs to a similar extent. In contrast, inability of muscle cells to secrete IL-1&bgr; markedly reduces induction of angiogenic factors and impairs neovascularization by cell implantation. Implanted cells do not secret angiogenic factors sufficient for neovascularization but, instead, stimulate muscle cells to produce angiogenic factors, thereby promoting neovascularization in ischemic tissues. Further studies will allow us to develop more effective treatments for ischemic vascular disease.

Cellular senescence impairs circadian expression of clock genes in vitro and in vivo.

Circadian rhythms are regulated by a set of clock genes that form transcriptional feedback loops and generate circadian oscillation with a 24-hour cycle. Aging alters a broad spectrum of physiological, endocrine, and behavioral rhythms. Although recent evidence suggests that cellular aging contributes to various age-associated diseases, its effects on the circadian rhythms have not been examined. We report here that cellular senescence impairs circadian rhythmicity both in vitro and in vivo. Circadian expression of clock genes in serum-stimulated senescent cells was significantly weaker compared with that in young cells. Introduction of telomerase completely prevented this reduction of clock gene expression associated with senescence. Stimulation by serum activated the cAMP response element-binding protein, but the activation of this signaling pathway was significantly weaker in senescent cells. Treatment with activators of this pathway effectively restored the impaired clock gene expression of senescent cells. When young cells were implanted into young mice or old mice, the implanted cells were effectively entrained by the circadian rhythm of the recipients. In contrast, the entrainment of implanted senescent cells was markedly impaired. These results suggest that senescence decreases the ability of cells to transmit circadian signals to their clocks and that regulation of clock gene expression may be a novel strategy for the treatment of age-associated impairment of circadian rhythmicity.

Reduced Nitric Oxide Causes Age-Associated Impairment of Circadian Rhythmicity

Impairment of circadian rhythmicity in the elderly has been suggested to cause age-associated diseases such as atherosclerosis and hypertension. Endothelium-derived nitric oxide (NO) is a critical regulator of cardiovascular homeostasis, but its production declines with aging, thereby inducing vascular dysfunction. We show here that impaired circadian rhythmicity is related to a decrease of NO production with aging. Treatment with an NO donor significantly upregulated the promoter activity of the clock gene Period via the cAMP response element–dependent and the E-box enhancer element–dependent pathways. Both phosphorylation and S-nitrosylation by NO are involved in this upregulation. In aged animals, endothelial NO synthase activity was markedly decreased during the daytime, along with impairment of clock gene expression and the circadian variation in blood pressure. Treatment of aged animals with an NO donor significantly improved the impairments. Inhibition of NO synthase activity also led to impairment of clock gene expression and blood pressure rhythm. These results suggest that NO is a key regulator of the circadian clock in the cardiovascular system and may be a novel target for the treatment of age-associated alteration of circadian rhythms.

Akt-Induced Cellular Senescence: Implication for Human Disease

Reduction-of-function mutations in components of the insulin/insulin-like growth factor-1/Akt pathway have been shown to extend the lifespan in organisms ranging from yeast to mice. It has also been reported that activation of Akt induces proliferation and survival of mammalian cells, thereby promoting tumorigenesis. We have recently shown that Akt activity increases with cellular senescence and that inhibition of Akt extends the lifespan of primary cultured human endothelial cells. Constitutive activation of Akt promotes senescence-like arrest of cell growth via a p53/p21-dependent pathway, leading to endothelial dysfunction. This novel role of Akt in regulating the cellular lifespan may contribute to various human diseases including atherosclerosis and diabetes mellitus.

Application of Hematopoietic Cells to Therapeutic Angiogenesis

Despite considerable progress in the field of cardiovascular medicine and surgery, ischemic heart disease is still the leading cause of death in advanced countries. In this context, it is no wonder why therapeutic angiogenesis, a way to ameliorate ischemic tissue from suffering dysfunction by increasing new blood vessels, gains so much attention from both clinicians and patients. In this review, we will briefly go through a decade of history in therapeutic angiogenesis including unraveling of its mechanisms, results obtained from clinical trials, and lessons learned from earlier investigations. We will then focus on an emerging, yet rapidly evolving field of hematopoietic cell therapy. Recent excellent studies seem to have brought us to the place where we might save so many patients from burden of ischemia, we should be aware that there are some controversies, and sometimes misunderstandings, regarding how or why this treatment does actually work, and what better way should we explore in order to get the best of its efficacy. With these caveats in mind, we will investigate the works elucidating the mechanisms and clinical efficacies of hematopoietic cell therapy.